Low molecular weight RNA species from chromatin.

Several methods of preparing low molecular weight RNA from chick embryo chromatin have been examined. Traditional methods for dissociating chromatin utilizing high concentrations of salt (greater than 2 M) followed by high-speed centrifugation resulted in very low yields of RNA. Increased yields of RNA were obtained by treating chromatin at lower salt concentration (0.2-0.5 M). By using low salt extraction and sodium dodecyl sulfate-phenol deproteinization, six to eight low molecular weight homogeneous RNA species were isolated from chick embryo chromatin and mouse myeloma chromatin. In the myeloma system, all these RNAs are metabolically stable. Each component is homogeneous as examined by gel electrophoresis and hybridizes with mouse DNA at a rate consistent with a single species. There are multiple gene copies for these RNA species in the mouse genome, varying from 100 to 2000 copies for the different species. One of these RNAs is identical with 5S rRNA. In addition, the redundancy of genes for 18S, 28S, and 5S rRNA and tRNA was determined. Approximately 300 copies for 18 and 28S rTRNA and 500 copies for 5S rRNA were found. tRNAs were on an average 110-fold redundant with about 55 different species measured.     

Release of template restriction in chromatin by nuclear 4.5s RNA.

The stimulatory mechanism of RNA synthesis of calf-thymus chromatin by nuclear 4.5 S RNA from the homologous tissue was investigated by using exogenously added Escherichia coli RNA polymerase. The RNA synthesis was initiated at low concentration of salt, and then the chain elongation was achieved at high concentration of ammonium sulfate in the presence of polyvinyl sulfate. Under these conditions the number of binding sites of RNA polymerase on chromatin which were capable of initiating RNA chain was increased by the addition of the 4.5 S RNA. This stimulation was presumed to result from the release of template restriction in chromatin. The polyvinyl salt minimized ribonuclease activity without changing the RNA polymerase activity bound to the template. Neither rearrangement nor release of chromatin proteins affected the amount or size of RNA produced. Preliminary analysis suggested that the molecular species of RNA produced upon the addition of the 4.5 S RNA from various tissues seemed to be heterologous.     

Escherichia coli 4.5S RNA gene function can be complemented by heterologous bacterial RNA genes.

The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.     

Correlation of RNA binding affinity of avian oncornavirus p19 proteins with the extent of processing of virus genome RNA in cells.

We purified the p19 proteins from the Prague C strain of Rous sarcoma virus, avian myeloblastosis virus, B77 sarcoma virus, myeloblastosis-associated virus-2(0), and PR-E 95-C virus and measured their binding affinities for 60S viral RNA by the nitrocellulose filter binding technique. The apparent association constants of the p19 proteins from Rous sarcoma virus Prague C, avian myeloblastosis virus, and B77 sarcoma virus for homologous and heterologous 60S RNAs were similar (1.5 x 10(11) to 2.6 x 10(11) liters/mol), whereas those of myeloblastosis-associated virus-2(0) and PR-E 95-C virus were 10-fold lower. The sizes and relative amounts of the virus-specific polyadenylic acid-containing RNAs in the cytoplasms of cells infected with Rous sarcoma virus Prague C, myeloblastosis-associated virus-2(0), and PR-E 95-C virus were determined by fractionating the RNAs on agarose gels containing methylmercury hydroxide, transferring them to diazobenzyloxymethyl paper and hybridizing them to a 70-nucleotide complementary DNA probe. In cells infected with Rous sarcoma virus Prague C we detected 3.4 x 10(6)-, 1.9 x 10(6)-, and 1.1 x 10(6)-dalton RNAs, in PR-E 95-C virus-infected cells we detected 3.4 x 10(6)-, 1.9 x 10(6)- and 0.7 x 10(6)-dalton RNAs, and in cells infected with myeloblastosis-associated virus-2(0) we detected 3 x 10(6)- and 1.3 x 10(6)-dalton RNAs. Each of these RNA species contained RNA sequences derived from the 5' terminus of genome-length RNA, as evidenced by hybridization with the 5' 70-nucleotide complementary DNA. The ratios of subgenomic mRNA's to genome-length RNAs in cells infected with myeloblastosis-associated virus-2(0) and PR-E 95-C virus were three- to five-fold higher than the ratio in cells infected with Rous sarcoma virus Prague C. These results suggest that more processing of viral RNA in infected cells is correlated with lower binding affinities of the p19 protein for viral RNA, and they are consistent with the hypothesis that the p19 protein controls processing of viral RNA in cells.     

A novel variety of 4.5 S RNA from Codium fragile chloroplasts

An unusual new chloroplast RNA has been isolated and sequenced in the siphonous green alga, Codium fragile. This RNA is 94 nucleotides in length, has an unusually high A + U content (73%), contains no modified residues, and is as abundant as a single chloroplast tRNA species. Although this RNA is 4.5 S in size, it bears little sequence homology to the widely found and highly conserved 4.5 S RNAs present in the chloroplasts of higher plants. Nevertheless, this RNA may indeed by analogous to the higher plant 4.5 S RNAs, since the Codium 4.5 S RNA has the potential to form a secondary structure which in many respects is remarkably similar to that of known chloroplast 4.5 S RNAs, and hybridization data strongly suggests that the 4.5 S RNA is part of the ribosomal RNA operon, as is the case in higher plant chloroplasts.     

Mouse nucleolin binds to 4.5S RNAh, a small noncoding RNA

4.5S RNAh is a rodent-specific small noncoding RNA that exhibits extensive homology to the B1 short interspersed element. Although 4.5S RNAh is known to associate with cellular poly(A)-terminated RNAs and retroviral genomic RNAs, its function remains unclear. In this study, we analyzed 4.5S RNAh-binding proteins in mouse nuclear extracts using gel mobility shift and RNA-protein UV cross-linking assays. We found that at least nine distinct polypeptides (p170, p110, p93, p70, p48, p40, p34, p20, and p16.5) specifically interacted with 4.5S RNAhin vitro. Using anti-La antibody, p48 was identified as mouse La protein. To identify the other 4.5S RNAh-binding proteins, we performed expression cloning from a mouse cDNA library and obtained cDNA clones derived from nucleolin mRNA. We identified p110 as nucleolin using nucleolin-specific antibodies. UV cross-linking analysis using various deletion mutants of nucleolin indicated that the third of four tandem RNA recognition motifs is a major determinant for 4.5S RNAh recognition. Immunoprecipitation of nucleolin from the subcellular fractions of mouse cell extracts revealed that a portion of the endogenous 4.5S RNAh was associated with nucleolin and that this complex was located in both the nucleoplasm and nucleolus.     

Small RNA molecules related to the Alu family of repetitive DNA sequences.

A rodent 4.5S RNA molecule with extensive homology to the Alu family of interspersed repetitive DNA sequences has been found physically associated with polyadenylated nuclear and cytoplasmic RNAs (W. Jelinek and L. Leinwand, Cell 15:205-214, 1978; S. Haynes et al., Mol. Cell. Biol. 1:573-583, 1981). In this report, we describe a 4.5S RNA molecule in rat cells whose RNase fingerprints are identical to those of the equivalent mouse molecule. We show that the rat 4.5S RNA is part of a small family of RNA molecules, all sharing sequence homology to the Alu family of DNA sequences. These RNAs are synthesized by RNA polymerase III and are developmentally regulated and short-lived in the cytoplasm. Of this family of small RNAs, only the 4.5S RNA is found associated with polyadenylated RNA.     

Use of recombinant plasmids to characterize collagen RNAs in normal and transformed chick embryo fibroblasts.

Two recombinant plasmids containing chick collagen DNA sequences have been used to characterize messenger RNAs for pro-alpha1 (type I) and pro-alpha2 collagen. Poly(A)-containing RNA from chick embryo calvaria and long bones, tissues which are very active in collagen synthesis, were electrophoresed on agarose gels containing methylmercuric hydroxide and transferred to diazobenzyloxymethyl paper; these covalently bound RNAs were hybridized to 32P-labeled pro-alpha1 or pro-alpha2 collagen DNA sequences derived from the recombinant plasmids. The pro-alpha1 collagen probe identified two RNAs, a major species of 5000 bases and a minor species of 7100 bases; the pro-alpha2 collagen probe hybridized to a major species very similar in size to the pro-alpha1 mRNA, about 5200 bases, and a minor species of 5700 bases. It is possible that the 7100 and 5700 base RNAs represent precursors of pro-alpha1 and pro-alpha2 collagen mRNA, respectively. When similar hybridization experiments were performed with RNA from chick embryo fibroblasts, both the pro-alpha1 and pro-alpha2 collagen mRNAs were observed, as well as their corresponding larger species. With RNAs from fibroblasts transformed by Rous sarcoma virus, however, the levels of all RNA species which hybridized with the pro-alpha1 and pro-alpha2 collagen DNA probes were significantly reduced.     

Fine structure of ribosomal RNA: I. Conservation of homologous regions within ribosomal RNA of eukaryotes

By molecular hybridization experiments the homologies between ribosomal RNAs from a unicellular organism (Gyrodinium cohnii), three invertebrates (Drosophila hydei, Chironomus thummi, Sciara coprophila), an amphibian (Xenopus laevis), and a mammal (mouse) were determined. Competition hybridization experiments demonstrated that portions of these homologous regions are the same in all the ribosomal RNAs tested, regardless of animal species. This conclusion based on hybridization data was confirmed by comparative fingerprint analysis. The ribosomal RNA sequences involved in heterologous hybridization have a higher A + T composition than the bulk ribosomal RNA. It appears from competition experiments of a heterologous hybridization that two thirds of the conserved similar regions are present in 18 S ribosomal RNA, and the remaining one third in 28 S ribosomal RNA. It is argued that these similar regions have been conserved during evolution due to their structural and/or functional role in ribosomal RNA.     

Computer analysis of the sequence relationships among 4.5S RNA molecular species from various sources

Nucleotide sequence homology among 4.5S RNAs from various organisms was examined by computer analysis to evaluate their sequence relationships. Chloroplast 4.5S rRNAs of wheat and tobacco were not significantly related to Escherichia coli 4.5S RNA, but were closely related to the 3'-terminus of bacterial 23S rRNA. Significant sequence homology was found between rat Novikoff hepatoma 4.5S RNAI and mouse and hamster 4.5S RNAs, suggesting that these RNAs are products of a family of genes with diverged sequences. E. coli 4.5S RNA had no significant sequence homology with any rodent 4.5S RNAs as a whole sequence. The E. coli, mouse and hamster 4.5S RNAs, however, were found to share a homologous 14-nucleotide sequence at the center of the molecules, which is known to exist as a conserved sequence in both Alu and Alu-equivalent sequences of mammalian DNAs.     

Cloning and characterisation of the abundant cytoplasmic 7S RNA from mouse cells.

A cDNA library has been prepared from mouse embryo small RNAs and screened for the presence of clones complementary to the highly abundant cytoplasmic 7S RNA. One clone (pA6) was selected which hybridized exclusively with 7S RNA on a Northern blot prepared from cytoplasmic RNA run on high resolution polyacrylamide/urea gels. Sequence analysis of this clone has shown that at least 65 nucleotides at the 5' end of 7S RNA are extensively homologous with the highly repeated mouse B1 family. Heterologous hybridisations between the cloned mouse 7S sequence and RNAs prepared from rat, human and chick cells have shown that the non-B1 part of the 7S RNA molecule has been highly conserved during recent eucaryotic evolution. There are multiple copies of 7S RNA genes in the genomes of mouse, human, rat and chick cells, but substantial differences exist in copy number and genomic organisation in these organisms.     

Nucleotide sequences of the 4.5 S and 5 S ribosomal RNA genes from tobacco chloroplasts

Summary The DNA sequences of the 4.5 S and 5 S RNA genes from tobacco chloroplasts have been determined. The coding regions for the mature 4.5 S and 5 S RNAs were identified by sequencing these RNAs. The 4.5 S and 5 S RNA genes are composed of 103 and 121 base pairs respectively. These two genes are separated by the 256 base pair spacer. Several unique features in the spacer and in the region downstream from the 5 S coding region are discussed.     

Mason-Pfizer virus RNA genome: relationship to the RNA of morphologically similar isolates and other oncornaviruses.

The 60-70S RNA of Mason-Pfizer virus (MPV) was iodinated in vitro and used in both direct and competitive molecular hybridization studies. MPV proviral sequences are present at a frequency of approximately one to two copies per haploid genome in the DNA of experimentally infected human cells. By nucleic acid competition hybridization, MPV RNA was found to be indistinguishable from the RNA of a virus (X381) isolated from a rhesus mammary gland and from RNA isolated from the cytoplasm of AO cells (Parks et al., 1973) and HeLa cells (Gelderblom et al., 1974), both previously reported to produce MPV-related particles. No homology was observed, however, between MPV RNA and the RNA, or the DNA, from two clones of HeLa cells obtained from the American Type Culture Collection. Hybridization of MPV 60-70S RNA to the DNA of normal tissues of humans and to the DNA of 11 other species revealed that MPV is not an endogenous virus of any of these species. Competition hybridization revealed no detectable sequence homology between the RNA of MPV and the RNAs of simian sarcoma virus, murine mammary tumor virus, murine leukemia virus, BUdR-induced guinea pig virus, or avian myeloblastosis virus. These nucleic acid studies substantiate previous ultrastructural and immunological findings that MPV and morphologically similar isolates constitute a distinct group of oncornavirus.     

Non-histone chromosomal proteins easily extractible from chick erythrocytes.

Those non-histone chromosomal proteins which are easily extractible from chick erythrocytes differ substantially from proteins similarly extracted from other tissues of various species. Although a protein P1 was isolated along with histone H1 by extraction of calf thymus chromatin with HC1O4, the same procedure did not extract this protein from chick erythrocyte chromatin of either normal or regenerating blood. Likewise , non-histone proteins extracted with 0.35 M NaCl from calf thymus differed from those of normal chick erythrocytes, which were qualitatively identical but quantitatively inferior to those of regenerating blood. The major protein of about 25 000 molecular weight, totally extracted with 0.35 M NaCl from calf thymus, was not found in chick erythrocyte chromatin, but rather another major protein of about 35 000 molecular weight was partially extracted from erythrocytes.